The OP9-DL1 System: Generation of T-Lymphocytes from Embryonic or Hematopoietic Stem Cells In VitroRoxanne Holmes and Juan Carlos Zúñiga-Pflücker1

Sunnybrook Research Institute and Department of Immunology, University of Toronto, Toronto, Ontario M4N 3M5, Canada

1 Corresponding author (jczp@sri.utoronto.ca )

INTRODUCTION

Differentiation of mouse embryonic stem cells (ESCs) or hematopoietic stem cells (HSCs) from fetal liver or bone marrow into T-lymphocytes can be achieved in vitro with the support of OP9-DL1 cells, a bone-marrow-derived stromal cell line that ectopically expresses the Notch ligand, Delta-like 1 (Dll1). This approach provides a simple, versatile, and efficient culture system that allows for the commitment, differentiation, and proliferation of T-lineage cells from different sources of stem cells. This article contains a series of protocols, the first of which describes the establishment, maintenance, and storage of OP9 and OP9-DL1 cells. Subsequent protocols detail how to co-culture the OP9 and OP9-DL1 cells with either ESCs or HSCs from fetal liver or bone marrow, leading to in vitro differentiation of the stem cells into lymphocytes.

RELATED INFORMATION

Preparation of the OP9 and OP9-DL1 cells should be started ~1 wk prior to initiating co-cultures with ESCs or HSCs. Protocols 3 and 4 use HSCs isolated from murine fetal liver or bone marrow. HSC isolation protocols are widely available (Klug and Jordan 2002 ; Schmitt et al. 2004 ; de Pooter et al. 2006 ; Bunting 2008 ). For a protocol to isolate and maintain mouse embryo fibroblasts (mEFs), see Preparing Mouse Embryo Fibroblasts (Nagy et al. 2006a ). For a protocol to derive or maintain ESCs, see De Novo Isolation of Embryonic Stem (ES) Cell Lines from Blastocysts (Nagy et al. 2006b ).

MATERIALS

Reagents

Anti-CD24 monoclonal antibody (mAb) (J11d clone) (for Protocol 3)

Use either culture supernatant that contains anti-CD24 mAb or purified anti-CD24 mAb (see Step 55).

BDPharmLyse (red blood cell lysing reagent; BD Biosciences 555899) (for Protocol 4)

Buffer for cell staining and cell sorting (for Protocol 4; see Step 72)

Complement, reconstituted from rabbit (Cedarlane CL3331) (for Protocol 3)

ESC medium (for Protocols 1, 2)

Freezing medium for OP9 cells (for Protocols 1, 2)

Leukemia inhibitory factor (LIF; 10 µg/mL; Chemicon LIF2010) (for Protocol 2; see Step 20)

Lympholyte-M (Cedarlane CL5120) (for Protocol 3)

Mice, 4-8 wk old (for Protocol 4; see Step 69)

Mice, fetal, at embryonic day 13-15 (for Protocol 3; see Step 52)

Mouse embryonic fibroblast (mEF) cells, irradiated and growing in a 6-cm dish (for Protocol 2)

Alternatively, a gelatin-coated 6-cm dish can be used. For a protocol to isolate and maintain mEFs, see Preparing Mouse Embryo Fibroblasts (Nagy et al. 2006a ).

Mouse embryonic stem cells (ESCs), stored frozen in liquid nitrogen (for Protocol 2)

R1 ESCs can be obtained from ATCC (SCRC-1036). For a protocol to derive or maintain ESCs, see De Novo Isolation of Embryonic Stem (ES) Cell Lines from Blastocysts (Nagy et al. 2006b ).

OP9 or OP9-DL1 cells, stored frozen in liquid nitrogen

OP9 cells can be obtained from the Riken Laboratory Cell Repository (Japan), and then transduced with a retroviral construct encoding Delta-like-1 to generate OP9-DL1 cells (Schmitt and Zúñiga-Pflücker 2002 ), or they can be requested from the Zúñiga-Pflücker laboratory.

OP9-DL1 or OP9 cells growing on a 6-well plate (for Protocol 2; see Step 39)

OP9-DL1 cells are used to generate T-lineage cells, and OP9 cells are used to generate B-lineage and myelo-erythroid cells.

OP9 medium

Phosphate-buffered saline (PBS; Hyclone SH30256)

Recombinant human Flt-3/Flk-2 ligand (R&D Systems 308-FK) (for Protocols 2-4)

Recombinant murine IL-7 (Peprotech 217-17) (for Protocols 2-4)

Trypsin solution, 0.25% (Invitrogen 15090046) (for Protocols 1, 2)

Dilute stock trypsin to 0.25% with PBS.

Equipment

Biosafety cabinet

Cell strainers (40-µm pore size; BD Falcon 352340) (for Protocols 2-4)

Centrifuge

Flow cytometer (for Protocols 2-4)

Forceps, 4-in. straight tips (Electron Microscopy Sciences 72991-4S) (for Protocol 4)

Forceps, curved fine points Dumont #7 (Electron Microscopy Sciences, 72800-D), and super fine points Dumont #5 (Electron Microscopy Sciences, 72700-D) (for Protocol 3)

Glass stopper, No. 24 (for Protocol 4)

Incubator, humidified (37°C and 5% CO2 )

Liquid-nitrogen cell storage system

Magnetic assisted cell sorter (MACS) (optional; see Protocol 4, Step 73)

Microscope

Plunger from a 3-mL syringe (for Protocol 3)

Scissors, dissecting (Electron Microscopy Sciences 72940) (for Protocols 3, 4)

Tissue culture plasticware

Carry out all procedures using standard aseptic technique with sterile plasticware (tissue-culture-treated 10-cm and 6-well plates, 15-mL and 50-mL centrifuge tubes, 2-mL cryovials, serological pipettes, and pipette filtered tips).

Water bath pre-set to 37°C (for Protocols 1, 2)

METHOD

Protocol 1: Preparation of OP9 Cells for Co-culture

Thawing OP9 or OP9-DL1 Cells

1. Add 12 mL of OP9 medium to a 15-mL centrifuge tube. 2. Thaw OP9 or OP9-DL1 cells quickly in a 37°C water bath. 3. Pipette the cell suspension slowly and gently from the cryovial tube, and transfer the contents to the 15-mL tube containing medium. 4. Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C. Resuspend the cells in 10 mL of OP9 medium. 5. Transfer the resuspended cells into a 10-cm dish, and place the dish in an incubator.

Maintaining OP9 or OP9-DL1 Cells

6. To passage the cells, remove the medium from the 10-cm dish. The cells will become confluent in the dish within 2-3 d, depending on the method used to freeze the cells. Passage the cells before they reach ~80% confluency ( Fig. 1 ).

7. Wash the plate with 4 mL of PBS. Discard the PBS. 8. Trypsinize the cells with 4 mL of 0.25% trypsin solution, and incubate the cells for 5 min at 37°C. 9. Disaggregate the cells from the dish by pipetting them up and down, and transfer the cell suspension into a 15-mL tube containing 4 mL of OP9 medium. 10. Wash the plate with PBS to remove any remaining cells, and transfer these cells to the same 15-mL tube. 11. Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C, and resuspend them in 4 mL of OP9 medium. 12. Transfer 1-mL aliquots of cells to four 10-cm dish each containing 9 mL of OP9 medium. Maintain the cells by splitting them at a ratio of 1-to-4, and passaging the cells every 2 d. Do not keep the cells in continuous culture for more than 6 wk. The cells can be used in subsequent protocols; e.g., Steps 28, 59, and 74.

Freezing OP9 or OP9-DL1 Cells

13. Passage the cells as described in Steps 6-11, except resuspend the cells (~8-10 x 105 cells), in 2 mL of freezing medium for OP9 cells per 10-cm dish. Preferably, freeze cells within the first two to three passages. 14. Aliquot 1 mL of cell suspension per cryovial. 15. Freeze the cells at -80°C, and then transfer them to liquid nitrogen for storage.

Protocol 2: In Vitro Generation of T-Lymphocytes from ESCs

Thawing ESCs

16. Prepare a 15-mL tube containing 12 mL of ESC medium. 17. Thaw the ESCs quickly in a 37°C water bath, and transfer the thawed cells slowly into the 12 mL of ESC medium. 18. Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C, and resuspend in 3 mL of ESC medium. 19. Seed ESCs onto a 6-cm dish containing irradiated mEF cells or a gelatin-coated 6-cm dish. 20. To keep the ESCs from differentiating, add 10 ng/mL of LIF when grown on mEF cells or 20 ng/mL of LIF when grown on gelatin. The mEF cells can be irradiated up to 2 d before using them as feeder cells. It is important that the mEF cell layer completely covers the surface of the tissue culture dish, because ESCs will begin to undergo differentiation within mEF cell-free areas.

Maintaining ESCs

21. The following day, change the ESC medium and again add the appropriate concentration of LIF. 22. Passage the ESCs the next day with trypsin-mediated disaggregation. Trypsin-mediated passage is used to break up large ESC colonies, allowing the culture to expand. i. Remove the medium from the 6-cm dish. Wash the plate with 3 mL of PBS. Discard the PBS. ii. Trypsinize the cells with 4 mL of 0.25% trypsin solution, and incubate the cells at 37°C for 5 min. iii. Disaggregate the cells from the dish by pipetting them up and down, and transfer the cell suspension into a 15-mL tube containing 3 mL of ESC medium. iv. Wash the plate with PBS to remove any remaining cells, and transfer these cells to the same 15-mL tube. v. Centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C, and resuspend them in 3 mL of ESC medium. 23. Seed ESCs in 3 mL of ESC medium onto irradiated mEF cells or gelatin-coated plates, and add LIF. 24. Repeat Steps 22 and 23 until the ESCs are needed for co-culturing. The ESCs grow as colonies. If they become too crowded, passage the cells more sparsely ( Fig. 1a ).

Freezing ESCs

25. Passage the ESCs as described in Step 22. Resuspend the cells in 3 mL of freezing medium for OP9 cells. 26. Aliquot 1 mL of cell suspension per cryovial (~3-6 x 105 cells/cryovial). 27. Freeze the cells at -80°C, and then transfer them to liquid nitrogen for storage.

Establishing ESC/OP9-DL1 Cell Co-cultures

Day 0: Initiation of Co-culture See Figure 2 for a schematic of the ESC/OP9 co-culture.

28. For each 10-cm dish containing 80%-90% confluent OP9 cells, remove the medium and replace it with 9 mL of fresh OP9 medium. The OP9 cells were prepared in Steps 1-12. 29. Harvest ESCs as a single cell suspension by trypsin-mediated disaggregation (Step 22). Resuspend the cells to a concentration of 5 x 104 cells per mL of OP9 medium. 30. Seed 1 mL of ESCs per 10-cm dish of OP9 cells, and return the dishes to the incubator (Fig. 1c ). Use OP9 cells (not ectopically expressing Dll1) to initiate the culture, because a more robust population of mesoderm colonies grows on these stromal cells as opposed to OP9-DL1 cells. The cells will be transferred onto OP9-DL1 cells at day 8 to induce T-lineage differentiation ( Fig. 2 ). See Troubleshooting.

Day 3: Medium Change

31. Remove the old medium, replace it with 10 mL of OP9 medium, and return cells to the incubator. The ESCs should have less defined borders and should start forming mesoderm colonies.

Day 5: Trypsin-Mediated Passage and Pre-plating

32. Remove the medium and trypsinize the cells (see Steps 7 and 8). Mesoderm colonies should be visible ( Fig. 1d ,e), and some colonies will appear three-dimensional with a wagon wheel shape, whereas others will appear like small cells clustered in a pile or as bunches. These colonies will become visible without the aid of a microscope and will appear as white circles in the dish. 33. Resuspend the trypsin-disaggregated cells with 4 mL of OP9 medium. 34. Return the dish to the incubator for 30 min. This pre-plating step will allow OP9 cells to readhere to the dish, which will limit the number of OP9 cells that are transferred. 35. Wash the non-adherent cells off the dish, filter the cells through a 40-µm cell strainer, and centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C. 36. Resuspend the cells in OP9 medium, seed 5 x 105 cells per 10-cm dish containing ~80% confluent OP9 cells in 10 mL of OP9 medium, and add 5 ng/mL of recombinant human Flt-3L. Return the dish to the incubator. The number of 10-cm dishes to be seeded should be equal to, or greater than, the number of analysis time points required. This ensures that the cultures remain undisturbed and there are enough samples for analysis (e.g., flow cytometry). Each 10-cm dish at day 5 yields enough cells to seed six to eight 10-cm dishes.

Day 8: Harvesting Semiadherent Hematopoietic Cells

37. Using the medium that is in the dish, wash the cells gently enough to keep the OP9 cell monolayer intact, but use enough force to remove the semiadherent cells. Clusters of round, shiny cells should be visible as small groups, either semiattached to the OP9 cells or in suspension ( Fig. 1f ). Check the dish under the microscope to ensure that all the round, shiny cells have been removed. See Troubleshooting. 38. Filter the cells through a 40-µm cell strainer, and centrifuge at 400g (1500 rpm) for 5 min at 4°C. 39. Resuspend the cells in 3 mL of OP9 medium per 10-cm dish initially seeded on day 5, and seed 3 mL per well of a 6-well plate containing either OP9-DL1 cells to generate T-lineage cells or OP9 cells to generate B-lineage and myelo-erythroid cells. 40. Add cytokines to each well; final concentrations are 5 ng/mL Flt-3L and 1 ng/mL.

Day 10: Medium Change

41. Remove the medium carefully so as to not disturb the co-culture. Large clusters of round, shiny cells should be present, with some cells detached in suspension. Collect all of the medium (which may contain differentiating lymphocytes) and centrifuge these cells at 400 g (1500 rpm) for 5 min at 4°C, thus limiting cell loss during the medium change. 42. Centrifuge the medium at 400g (1500 rpm) for 5 min at 4°C. Resuspend the cells present in the spent medium with 3 mL of OP9 medium containing 5 ng/mL Flt-3L and 1 ng/mL IL-7. 43. Transfer the medium and cells to the same well, and return the plates to the incubator.

Day 12: No-Trypsin Passage

44. Disaggregate the cells without the use of trypsin by simply pipetting them up and down to create a cell suspension. Large clusters of round, shiny cells should be present throughout the dish, with some cells detached in suspension ( Fig. 1g ). The OP9 cells may lift as a sheet, which will be broken up by forceful pipetting. 45. Filter the cells through a 40-µm cell strainer. 46. Wash the well with 3 mL of PBS, filter the suspension through the same cell strainer, and centrifuge the cells at 400g (1500 rpm) for 5 min at 4°C. 47. Resuspend the cells in 3 mL of OP9 medium, and seed the cells onto a new well of OP9 or OP9-DL1 cells, with the addition of 5 ng/mL Flt-3L and 1 ng/mL IL-7. Return the plates to the incubator. This is a good time point to analyze by flow cytometry to determine the presence of different lympho-hematopoietic cell populations. As shown in Figure 3 , for ESCs cultured on OP9 cells, the majority of cells will be erythro-myeloid Ter119 + or CD11b + , while for ESCs cultured on OP9-DL1 cells, most of the cells will resemble DN1 (CD44 + CD25- ) stage thymocytes, although some DN2 (CD44 + CD25 + ) and DN3 (CD44- CD25 + ) stage cells may be present.